A number of motility disorders are believed to be caused by defects in the neural control of the gastrointestinal (GI) tract. However, the underlying pathophysiology remains largely undefined and often the treatment is ineffectively targeted. Our long-term goal is to develop methods for the prevention and treatment of conditions qualified as neuropathies based on understanding of the inhibitory purinergic signaling in the gut. The central hypothesis of this proposal is that beta-nicotinamide adenine dinucleotide ((3-NAD) is a novel inhibitory neurotransmitter in the GI tract. Particularly, we hypothesize that (3-NAD is stored in synaptic vesicles, is released upon action potential firing, activates P2Y purinergic receptors and apamin-sensitive small conductance Ca2+-activated potassium (SK) channels on either interstitial cells of Cajal (ICC), smooth muscle cells (SMC) or fibroblast-like cells (FLC), causes membrane hyperpolarization and smooth muscle relaxation, and is removed by CD38- and CD157-mediated metabolism and by uptake in nerve varicosities. The work, carried out with colon preparations from humans, non-human primates, and mice, will: (i) determine the cell types that are the primary source of release of (3-NAD and ATP during EPS of enteric nerves (Aim 1), (ii) examine whether the expression, distribution, and function of vesicular nucleotide transporter (VNUT) is consistent with the role of (3-NAD as an inhibitory motor neurotransmitter in GI muscles (Aim 2), (iii) determine the primary postjunctional targets of PNAD action (Aim 3), and (iv) examine the major mechanisms of removal of (3-NAD (i.e., enzymatic degradation or neuronal uptake), and hence of terminating its neurotransmitter actions (Aim 4). We will examine overflow of purine neurotransmitters and their metabolites using high performance liquid chromatography (HPLC) techniques along with immunohistochemistry, protein biochemistry, molecular biology techniques, electron microscopy, flow cystometry analysis, electrophysiology, and functional approaches to better understand the role of p-NAD and other purines as putative neurotransmitters. This research has the potential to fundamentally advance our understanding of enteric purinergic signaling, and could have important implications for developing novel therapeutic strategies for GI motility disorders based on defects of neural control of the GI muscles.